Production of Monoclonal Antibody for Identification of Tomato Zonate Spot Virus

[[abstract]]The genus Tospovirus belongs to the family Bunyaviridae and possesses quasi-spherical enveloped particles of 80-120 nm in diameter containing a large genome consisting of three RNA segments, denoted S, M, and L according to their sizes. Based on the serological and phylogenetic relationships of the S RNA-encoded nucleocapsid proteins (NPs), 28 tospovirus spices have been characterized so for. Tomato zonate spot virus (TZSV), a member of Tospovirus genus, was first observed on tomatoes and chili peppers during a field survey in Yunnan Province, China in 2005. TZSV caused concentric zoned ringspots on ripe fruits and necrotic lesions on leaves of diseased tomato plants. Genome sequencing of TZSV revealed that it shares high NP amino acid (aa) identities of 80.9% and 81.7% with those of Calla lily chlorotic spot virus (CCSV) and Tomato necrotic spot virus (TNSV), respectively. CCSV was first discovered in Taiwan in 2000, and TNSV is a newly reported tospovirus collected from Guizhou Province, China in 2009. A previously produced monoclonal antibody (MAb) against the NP of CCSV (MAb-CCSV-N) positively reacted with TZSV in both indirect enzyme-linked immunosorbent assay and western blotting, indicating that this MAb can not distinguish TZSV and CCSV. To develop a fast and accurate tool for identification of TZSV, the NP of TZSV expressed by the bacterial pET expression system was used as an immunogen for preparation of TZSV-specific MAb (MAb-TZSV-S18D7H2), which can be successfully used to differentiate TZSV from CCSV. Epitope mapping indicated that the target epitope of MAb-TZSV-S18D7H2 is located at the aa 77-86 region (GHKIVASGAD) of NP. Moreover, the TZSV- and CCSV-specific primer pairs were designed and used in multiplex reverse transcription-polymerase chain reaction (RT-PCR) to simultaneously detect and differentiate TZSV and CCSV. Our efficient detection tools, including serological and multiplex RT-PCR methods, are valuable for quarantine and inspection of foreign TZSV and domestic CCSV